The crown of a human tooth has an outer layer of highly-mineralized tissue called enamel, beneath which is dentin, a less-mineralized tissue which forms the bulk of the tooth-crown and root. The composition and structure of enamel and dentin are different, resulting in different thermal properties. This gives an opportunity to characterize enamel and dentin from their thermal properties and to visually present the findings as a thermal map. The thermal properties of demineralized enamel and dentin may also be sufficiently different from sound tissue to be seen on a thermal map, underpinning future thermal assessment of caries. The primary aim of this novel study was to produce a thermal map of a sound, human tooth-slice to visually characterize enamel and dentin. The secondary aim was to map a human tooth-slice with demineralized enamel and dentin to consider future diagnostic potential of thermal maps for caries-detection. Two human slices of teeth, one sound and one demineralized from a natural carious lesion, were cooled on ice, then transferred to a hotplate at 30°C where the rewarming-sequence was captured by an infra-red thermal camera. Calculation of thermal diffusivity and thermal conductivity was undertaken, and two methods of data-processing used customized software to produce thermal maps from the thermal characteristic-time-to-relaxation and heat-exchange. The two types of thermal maps characterized enamel and dentin. In addition, sound and demineralized enamel and dentin were distinguishable within both maps. This supports thermal assessment of caries and requires further investigation on a whole tooth. The portion of tooth visible within the human mouth, known as the crown, has two main layers of mineralized tissue: enamel and dentin. Tooth enamel is produced by ameloblasts and has the highest mineral content of any tissue within the human body at approximately 96% mineral by weight, compared to dentin which is produced by odontoblasts at approximately 70% mineral by weight  has limited penetration depth of dental caries but can be improved by using longer wavelengths of near-infrared (780–1550 nm), especially 1,310 nm, due to enamel transparency at these wavelengths. Penetration-depth of optical coherence tomography (OCT) can also be limited but detection of lesions just beyond the dentin-enamel junction have been reported. Mineralized tooth-tissue possesses the ability to auto-fluoresce and quantitative light-induced fluorescence (QLF) uses this property, whereas DIAGNOdent (Kavo) and the LF-pen is thought to use fluorescence from protoporphyrin IX and associated bacterial products, not the mineralized tissue  than X-rays (0.01–10 nm), is non-ionizing and totally harmless. The Herschel family were central in discovering infra-red radiation and William Herschel was credited in 1800, and John Herschel produced the first Thermogram in 1840 Holst, . By the −7m2/s, dentin ≈1.83–2.6 × 10−7m2/s) and thermal conductivity , as shown in Table In Kaneko et al.  and ZakiA map provides spatial information from co-ordinates on two axes, such as X and Y. The use of a thermal value for each co-ordinate corresponding to a pixel of tooth-tissue could be represented from a grayscale, producing a thermal map specific to that tooth-tissue. This could show the spatial relationship of the thermal properties of the tooth-tissue as an image, rather than a series of numerical values. This may be more clearly understood from the map providing a 2-dimensional relationship of the thermal properties across the whole surface of the tooth-tissue. The thermal properties of enamel and dentin may be sufficiently different to visually distinguish enamel from dentin. Demineralized areas within both tissues may also have different thermal properties due to tissue-changes, such as mineral loss from caries, which may be seen in the thermal map.The primary aim of this study was to produce a thermal map of a sound, human tooth-slice to visually characterize human enamel and dentin. The secondary aim was to map a human tooth-slice with demineralized enamel and dentin to consider future diagnostic potential of thermal maps in caries-detection.Two lower-third molar teeth were ethically sourced from Leeds Dental Institute Research Tissue Bank. Both teeth were cut bucco-lingually with an Accutom-5  into 1 mm-thick tooth-slices and then polished with an 800-grit abrasive sheet, with the addition of distilled water as needed. Slice-thickness was measured with a digimatic micrometer (IP65 Quantumike Mitutoyo). Photographs and radiographs of each tooth-slice were taken.A purpose-built thermal cube provided a stable thermal environment at 22°C Figure  with macA fixed camera-mounting held the FLIR SC305 thermal camera with a x4 lens (resolution of 100 μm) at a focal distance of 8 cm. The parameters used with the camera were: Emissivity 0.96, Reflective Apparent Temperature 27.2°C and Humidity 50%. The recording-rate was 9-frames-per-second and data-collection was with ThermaCAM Researcher Professional 2.10 Software.An aluminum hotplate (Bibby Techne DB-2TC) was positioned within the cube and stabilized at 30°C. Thermocouples (Omega Type-K SA1XL) were attached to the 0.5 mm thermal pad  of the copper-based specimen-carrier.Each tooth-slice was paper-dried, placed on the thermal pad and cooled on a block of ice until a temperature of 2°C was recorded on a Fluke 52 II thermometer. The carrier was manually transferred to the aluminum hotplate.c), which required bespoke software for use in Matlab (MathWorks®).Data-capture by the thermal camera commenced prior to transfer of the carrier. Processing of data from selected areas-of-interest circled in blue Figure  occurredc) of the thermal pad and the tissue area-of-interest was applied to the formulae below to calculate the thermal diffusivity:The difference in value of characteristic-time-to-relaxation (τ−7m2/Sec)α = Thermal DiffusivitycDiff = τcTissue − τcThermal Pad (Seconds)τOnce the value of thermal diffusivity was known, thermal conductivity could be calculated:κ = Thermal Conductivity (W/mK)−7m2/Sec)α = Thermal Diffusivity(103) = 2800 for enamel and 2248 for dentin  = 710 for enamel and 1066.4 for dentin  software, from which two thermal maps were produced.The τThe thickness of the sound tooth-slice measured less than a millimeter in all areas, with the greatest enamel-thickness in the middle at 0.78 mm, the greatest crown-dentin-thickness recorded was on the left at 0.784 mm and the single measurement of the root-dentin was 0.785 mm. The carious tooth-slice was thicker in all areas, with the maximum thickness of enamel at 1.227 mm in the middle, similarly for crown-dentin at 1.223, and the thickest root-dentin was 1.166 mm on the left (see Table c) is used to calculate the thermal properties of diffusivity and conductivity in conjunction with tissue-thickness. The results indicate from the sound tooth-slice that enamel has a higher value of thermal diffusivity (3.79–4.15 × 10−7m2/s) and thermal conductivity (0.75–0.83 W/mK) than dentin thermal diffusivity (1.89–2.81 × 10−7m2/s) and thermal conductivity (0.45–0.67 W/mK) except for one outlier for root dentin with thermal diffusivity of 7.71 × 10−7m2/s and thermal conductivity of 1.85 W/mK. All values are provided in Table −7m2/s and dentin at 1.83–2.6 × 10−7m2/s and thermal conductivity of enamel at 0.45–0.93 W/mK and dentin at 0.11–0.96 W/mK, as previously quoted in Table The initial rewarming temperatures for the sound tooth-slice in both crown- and root-dentin (green and pink broken-lines) were lower than the two areas of enamel (purple and blue broken-lines). It took circa 30 s to reach thermal equilibrium of all tissues  and root-dentin (0.57 × 10−7m2/s) are reduced, compared to others' findings of 1.83–2.6 × 10−7m2/s. The carious area of dentin has an increased value of 1.01 × 10−7m2/s which is comparable to the carious area of the enamel (1.13 × 10−7m2/s). The thermal conductivity of carious enamel (0.22 W/mK) is similar to carious dentin (0.24 W/mK). All other values of thermal conductivity for enamel and dentin within the carious tooth-slice are lower than others' findings. All values are provided in Table Within the carious tooth-slice, the rate of rewarming in the two sound enamel areas-of-interest (purple and blue broken-lines) are similar, whereas the enamel carious lesion is slower (red solid-line) Figure . CariousThe two thermal maps distinguish the mineralized tissues of enamel, dentin and the carious areas of both tissues using the thermal properties of characteristic-time-to-relaxation and heat-exchange during rewarming Figure .Infra-red thermal imaging is a technique which is yet to be maximized within the field-of-medicine and its subsidiary specialty - dentistry. Published work for determining the thermal properties of tooth-tissue  which was confirmed with a USAF1951 Positive Test Target. OCT has a higher resolution at 5–15 μm (from ≈33 lp/mm), as does DiFOTI at 43 pixels/mm (≈ 21 lp/mm) (Lancaster et al., The use of thermal imaging to detect approximal caries is unlikely as it cannot penetrate tissues in the way X-rays do. However, detection of early smooth-surface lesions and occlusal lesions would allow preventive measures to be prescribed. X-rays have limitations, as previously mentioned, as do optical detection methods. Thermal imaging may complement our current armamentarium. Detection of active and arrested caries remains uninvestigated with thermal imaging and consideration will be needed for other potential causes of difference in tooth structure and composition, e.g., amelogenesis imperfecta and molar incisor hypomineralization.in-vitro from their thermal properties, as seen in the thermal maps of heat-exchange and characteristic-time-to-relaxation. The heat-exchange map produces better contrast between enamel and dentin than the characteristic-time-to-relaxation map. Within enamel and dentin, demineralized tissue can be detected in both maps, with heat-exchange providing the greatest contrast within both tissues. These thermal maps support further investigation of thermal imaging to complement diagnosis of caries.The enamel and dentin of tooth-slices can be characterized PL, DB, designed the Study, undertook the acquisition, analysis and interpretation of data, wrote the first draft of the manuscript, provided contribution to revision and final approval of the manuscript and are accountable for the work presented. FC, VC, were involved with conception of the design, revision and approval of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study sought to compare the root canal debridement ability of Neolix, Reciproc and ProTaper rotary systems in long oval-shaped root canals.Eighty five extracted single-rooted human teeth with long oval-shaped single root canals were selected and divided into three experimental groups(n=25) and one control group (n= 10). Root canals were filled with Vitapex radiopaque contrast medium and prepared with Neolix, Reciproc or ProTaper systems. The control group only received irrigation. Digital radiographs were obtained at baseline and postoperatively and subjected to digital subtraction. The percentage of reduction in contrast medium was quantified at 0-5 mm and 5-10 mm distances from the apex. The data were analyzed using one-way ANOVA and t-test.P=0.6). In the 5-10mm segment a significant difference was found in this regard among the ProTaper and Reciproc groups (P=0.02) and the highest mean percentage of contrast medium was removed by ProTaper. But, difference between ProTaper and Neolix as well as Neolix and Reciproc was not significant. In Neolix (P=0.024) and Reciproc (P=0.002) systems, the mean percentage of the contrast medium removed from the 0-5mm segment was significantly greater than that in 5-10mm segment; however, this difference was not significant in ProTaper group (P=0.069).The mean percentage of the contrast medium removed was not significantly different in the 0-5mm segment among the three groups ( Neolix single-file system may be a suitable alternative to ProTaper multiple-file system in debridement of long oval shaped canals. Key words:Root Canal Preparation, Debridement, Root Canal Therapy. Mechanical preparation of the root canal system (RCS) is a major step in achieving a successful endodontic treatment. Despite the technical advances in endodontics, root canal preparation is still highly influenced by the complex and variable anatomy of the RCS ,2. StudiRoot canals with maximum root canal diameter twice the minimum root canal diameter are referred to as oval-shaped canals. Oval-shaped root canals with maximum root canal diameter 2-4 times the minimum root canal diameter are referred to as long oval-shaped canals . Due to Introduction of nickel titanium (Ni-Ti) rotary instruments significantly enhanced mechanical preparation of the RCS .Considering the gap of information on the quality and efficacy of the newly introduced endodontic systems especially in oval-shaped root canals, this study aimed to compare the debridement efficacy of Neolix , Recirpoc  and conventional ProTaper  for preparation of long oval-shaped root canals.in vitro, experimental study was conducted on 85 single-rooted single-canal human teeth with long-oval shaped root canals. The teeth had been extracted for orthodontic reasons and periodontal problems. The research protocol was approved by the institutional Ethics Committee of the Vice Chancellor of Research, Hamadan University of Medical Sciences(Protocol No:IR.UMSHA.REC.1394.97). The teeth were randomly divided into three groups of 25 and one control group of 10. The inclusion criteria were as follows: (I) Freshly extracted teeth with a single long oval-shaped root canal  (II) Closed apex and (III) No cracks, curvature, anomaly, fracture, extensive caries, root resorption or previous endodontic treatment.This Access cavity was prepared using a #2 round bur and high speed hand piece; LA Axxess kit  was then used to standardize the access cavity walls; next, #2, 3 and 4 Gates Glidden drills  with low-speed hand piece were used for flaring of the coronal section of the root canal to 2-3mm below the cementoenamel junction (CEJ). Root canal was rinsed with 5mL of 5.25% sodium hypochlorite solution  using a 27 gage needle. A #10 K-file  was used to maintain root canal patency. The file was introduced into the canal until the file tip was seen at the apex. Of this length, 0.5mm was subtracted to obtain the wor-king length. Root canals were then filed with #15 and #20 K-files to obtain a glide path; 3mL of 5.25% sodium hypochlorite solution was used for rinsing of the root canal between filings. Final rinse was done with 3mL of sterile saline  and the root canals were dried with paper points . Next, the entire root canal space was filled with radiopaque contrast medium  using Vitapex microtip to assess the efficacy of root canal debridement . Then, an impression was taken from the position of the sensor, position of the tube and the two ends of the toothpick using putty impression material Fig. . All theRoot canal preparation with ProTaper rotary system(P):In the P group, root canals were prepared using ProTaper files. This system includes three files for shaping and three files for finishing. Shaping files include SX, S1 and S2 with ISO 19, 17 and 20 tip diameters. The finishing files include F1 and F2 with ISO 20 and 25 tip diameters. ProTaper files were used in the P group in the following order: S1 until resistance is met, SX until resistance is met, S1 to the working length, S2 to the working length, F1 to the working length and F2 to the working length. The files were used at 300 rpm speed and 5 N/Cm torque. An Endo IT electric motor  was used to control the speed and torque. Each file was used for flaring of four teeth.Root canal preparation with Reciproc rotary system(R):In the R group, a #20 K-file was first introduced to the root canal. Then, #25 Reciproc was activated in a reciprocating motion by VDW silver electric motor  and was gradually introduced to the canal with pecking motion with 3mm range and brushing motion. After three pecking motions, the instrument was removed and the root canal was rinsed with 3mL of saline. This procedure was repeated three times. A #10 K-file was repeatedly used to ensure root canal patency to the working length .Root canal preparation with Neolix single-file rotary system(N):In the N group, Endo IT electric motor was used to control the speed and torque; the speed was adjusted at 300-500 rpm and 1.5 N/Cm torque with pecking and brushing motions. First, C1 file was used to flare the coronal third and eliminate dentin barriers with brushing motion only in the coronal area. Next, A1 file was passively used for preparation of the middle and apical thirds. During filing and after three to four brushing motions, the root canals were rinsed with saline and patency was ensured using #15 K-file. Eventually, the file with pecking motion was used to the working length to complete shaping of root canal.Time of instrumentation in all groups was averagely four minutes. During filing, the root canals were filled with saline. The volume of irrigating solution was 20mL for each tooth. To prevent washout of radiopaque medium, sodium hypochlorite was not used. In the control group, root canals were rinsed with 20mL of saline but filing was not done . Pre- an-Statistical analysis:P<0.05 was considered statistically significant.Data were analyzed using SPSS version 19  and descriptive statistics. The Kolmogorov Smirnov test, one-way ANOVA, t-test and Tukey’s test were used for data analyses. P=0.6).The mean values were compared using one-way ANOVA and t-test. P=0.02) and the ProTaper group showed the highest mean percentage of contrast medium removed. The results of Tukey’s test revealed no significant difference between the Neolix and ProTaper (P=0.13) or Neolix and Reciproc (P=0.66) in 5-10mm segment; however, a significant difference was noted between the ProTaper and Reciproc groups in the mean values (P=0.01)( (P=0.01).P=0.024) and Reciproc (P=0.002) groups. In both mentioned groups, the mean percentage of the contrast medium removed from the 0-5mm segment was significantly greater than that in 5-10mm segment; however, this difference was not significant in ProTaper group (P=0.069).The mean percentage of the contrast medium removed from the 0-5mm segment was compared with that in 5-10mm segment in each group by t-test. Based on the results , signifiAside from the complex anatomy of root canals, limitations of preparation techniques are among the main reasons for inefficient cleaning of such root canals . In ordeet al., in 2013 ; this finding is in line with that of Ruckman et al., in 2013  revealed no significant difference  efficacy and optimal flexibility enabling the operator to have a suitable tactile sense while performing circumferential filing motion. This is particularly important for cleaning and shaping of oval-shaped root canals. On the other hand, Reciproc system is manufactured by M-Wire NiTi system, which, according to the manufacture, confers increased fatigue resistance and flexibility to the files. Considering all the above, it may be concluded that Neolix, due to higher flexibility, enables better contact with the buccal and lingual walls  with brushing motions and thus, has superior efficacy to Reciproc in preparation of long oval canals. Moreover, as stated earlier, previous studies ,13,15 haet al. (In our study, the prepared root canal areas were greater in 0-5mm segment compared to 5-10mm segment, which was similar to the results of Weiger et al. . The croIn general, none of the systems used in our study completely debrided the oval-shaped canals, which is in line with the findings of previous studies -3,4,16.Neolix single-file system may be a suitable alternative to ProTaper multiple-file system in debridement of long oval shaped canals.


The miR-199a-5p was found to be HIV-specific and expressed in all HIV+ groups as opposed to HIV– controls. Moreover, these are the first studies to reveal clearly the highly discriminatory miRNAs at the level of the disease state, cell type, and HIV-specific miRNAs.The potential involvement of host microRNAs (miRNAs) in HIV infection is well documented, and evidence suggests that HIV modulates and also dysregulates host miRNAs involved in maintaining the host innate immune system. Moreover, the dysregulation of host miRNAs by HIV also effectively interferes directly with the host gene expression. In this study, we have simultaneously evaluated the expression of host miRNAs in both CD4+ and CD8+ T-cells derived from HIV-positive (HIV+) individuals , therapy-naïve long-term non-progressors (LTNP), and HIV-negative (HIV–) healthy controls. miRNAs were run on Affymetrix V2 chips, and the differential expression between HIV+ and HIV− samples, along with intergroup comparisons, was derived using PARTEK software, using an FDR of 5% and an adjusted  MicroRNAs (miRNAs) are small 21–22 nt), non-coding RNA fragments found in many organisms, from plants to humans, which function to negatively regulate gene expression. In humans, roles for miRNA have been identified in metabolism, growth, and development. Dicer, a protein critical for miRNA processing, has been associated with proper development of the limbs, lungs, and hair follicles and in T-cell differentiation –22 nt, n. As negaet al. showed that the vast majority of host miRNAs are downregulated in HIV infection of peripheral blood mononuclear cells (PBMCs). When individuals were separated into groups based on their T-cell counts and viral loads (VLs), specific miRNA “profiles” could be seen for each of the classes. Furthermore, many miRNA changes in patient cells could not be accounted for by infection alone, indicating a complex role for miRNA in gene regulation [Viruses are known to subvert and manipulate the human gene machinery, and miRNAs are no exception. Host miRNAs are dysregulated by viruses, and both DNA and RNA viruses have been found to encode and express miRNAs in the infected host, demonstrating the importance of the role of host- and virus-encoded miRNAs in host-virus interactions. The miRNA expression profile of cells is altered in both clinical PBMC samples and cultured cells. Cells transfected with an infectious HIV-1 strain have significantly altered miRNA profiles compared to control cells . In 2008gulation .et al. showed overexpression of host miRNAs in resting T-cells that target sequences in the 3′ end of HIV-1 RNA, silencing viral mRNA and enforcing latency [et al. showed that PBMC miRNA profiles could distinguish elite suppressors (ES) and uninfected controls from viremic HIV-1 infected patients [et al. showed a set of 18 differentially expressed miRNAs, which could identify the outcome of HIV disease at the chronic stage more accurately. Six out of 18 miRNAs were significantly related to faster rate of CD4+ T-cell decline [In 2007, Huang  latency . Furtherpatients . Their r decline . StudiesSince all the studies to date have been performed on whole PBMC or tissue, we endeavored to address disease- and cell-type-specific miRNAs and their role in HIV pathogenesis. We have adopted a novel approach for this study, which simultaneously analyzes miRNAs from the CD4+ and CD8+ T-cells from viremic, aviremic BDL patients, and elite controllers. This study is unique in showing the HIV disease-stage and cell-type specificity of miRNA during HIV infection and its natural control in elite controllers.Patients were classed into disease groups based on their HIV plasma viral load (VL) and also the antiretroviral drug treatment, as shown in vs. HIV− analysis  available in the Partek Genomics Suite. Data in analysis , we examanalysis a–d. In cThe underlying reasons for this distinction between CD4+ and CD8+ T-cells in their ability to segregate HIV disease and non-disease groups remain unclear. PCA was also used to determine cell specificity in relation to disease group by directly comparing the CD4+ and CD8+ T-cells of each disease state. This analysis indicated a clear segregation of CD4+ and CD8+ T-cell type based on disease state, implying cell-specific functional segregation during HIV disease stages a–c and iFrequency histograms representative of the CD4+ and CD8+ T-cell datasets a,b show Upon a quality and integrity check using various algorithms discussed above, we analyzed differential miRNA expressions between disease groups and disease groups based on cell types. Heat maps, coupled with hierarchical clustering, were used throughout to visualize the validity and integrity of differential expressions between each contrast.p-values  were taken together and compared against the HIV– healthy donors using the false discovery rate (FDR). Two DE miRNAs were found specific to CD4+ T-cells (miR-199a-5p and miR-720) , and twop-values .When HIV-infected cells were compared to uninfected cells  using thp < 0.05, we also visualized the dataset using no FDR with p < 0.01. It is important to state that, when no FDR was applied to the analysis of differentially expressed miRNAs in infected vs. uninfected CD4+ and CD8+ T-cells, more miRNAs were observed, as expected. However, in this analysis, the top-ranked highly significant miRNAs that were observed in the FDR method were also fully covered in the non-FDR analysis. This dual-analysis served to ensure that statistical stringency did not occlude biologically relevant miRNAs , and three DE miRNAs were identified in CD4+ LTNPs   were individually compared against CD4+ T-cells from the HIV– controls using the FDR miR-126) . All of  changes . Interes changes .p < 0.01. This suggests that, although miR-199a-5p was differentially expressed in HIV infection in all three disease states, it was not expressed significantly enough to be picked up for CD4+ aviremic vs. HIV− comparison due to statistical stringency. Thus, taken together, the FDR ) indicate that miR-199a-5p was downregulated in the viremic state and that its upregulation appears to be characteristic of an HIV– state in healthy individuals —six downregulated and one upregulated. This upregulated miRNA, miR-345, demonstrated a 11.6-fold change with a p-value of 3.79 × 10−5 , and all three showed downregulation  were identified , all of which were supported by strong p-values  cells only.The main reason for these comparisons was to derive specificity of CD4+ or CD8+ T-cells in predicting a disease association. To do this, simultaneous comparison of the relationship between miRNA expression CD4+ and CD8+ T-cells in different disease groups for both cell types was carried out. For the first contrast, we found three DE miRNA for the CD4+ T-cells and five for the CD8+ T-cells during viremia. No overlapping miRNAs were seen. In contrast, for the LTNP CD4+ and CD8+ T-cells, four DE miRNAs in CD4+ T-cells and five in CD8+ T-cells during the non-progressive phase of the HIV disease were identified. The miRNA-199a-5p was found to be specific to the HIV+ CD4+ T-cells. Interestingly, miRNA-126 was the only miRNA found overlapping between CD4+ and CD8+ T-cells during the non-progressive HIV disease, which, according to the current literature, is the first miRNA possibly related to the non-progression of HIV disease b. No difvs. CD8+ T-cells, aviremic CD4+ vs. CD8+ T-cells, LTNP CD4+ vs. CD8+ T-cells, and negative CD4+ vs. CD8+ T-cells using the FDR method to identify DE miRNAs for each disease group. Using the FDR method and an adjusted p < 0.05, only one miRNA was differentially expressed. This miRNA was miR-151-5p, and was differentially expressed between the CD4+ and CD8+ T-cells of the LTNP group with a fold change of 10.26 and a p-value of 1.95 × 10−5, suggesting that it is a possible biomarker of non-progressive HIV disease based on both cell types.A direct comparison of CD4+ with CD8+ T-cells for the same disease group was carried out to obtain disease-stage-specific miRNAs common to both cell types. We performed comparisons between viremic CD4+ p < 0.01. By relaxing this statistical stringency, differential expression was observed for all contrasts , but was seen also in the aviremic groups only upon relaxing the FDR (p < 0.01).miRNA-199a-5p was significant and found to be specific to HIV+ CD4+ T-cells in viremic and LTNP groups using the FDR method  observed in qRT-PCR was consistent with the trend (FC-6.31). In the CD4+ T-cells—HIV+ vs. HIV− and LTNP vs. HIV negative comparisons—the qRT-PCR results also showed consistent trends of expression with the microarray. In the HIV+ vs. HIV− comparison, the microarray result of −5 fold downregulation was consistent with the qPCR result of −209. Again, in the LTNP vs. negative CD4+ T-cells, the downregulation detected in the microarray analysis (−7 fold) was also consistent in trend seen in qRT-PCR, where the detected fold change was −50 fold.The results of qRT-PCR for miR-199a-5p showed a consistent trend with the results of the microarray. In the CD4+ viremic vs. negative for miR-199a-5p. This comparison was significant for the microarray only in the non-FDR analysis. The fold change determined by qRT-PCR was −2.15 fold (data not shown), which is again in agreement with the trend of downregulation seen for this miRNA in CD4+ HIV-infected T-cells in the microarray.qRT-PCR analysis was also performed for the comparison CD4+ aviremic Along with their association with pathological and physiological pathways, host miRNAs play a significant role in many viral infections, including HIV. In this work, we describe the first direct examination of virus-mediated cellular miRNA expression manipulation in the CD4+ and CD8+ T-cell fractions taken from HIV+ patients at differing stages of HIV disease. This disease-stage stratification was primarily based on the detectable virus in the plasma of diseased individuals and the length of infection. The main rationale behind conducting a parallel analysis of CD4+ and CD8+ T-cells for miRNAome was based on the roles of these cells in HIV infection. CD4+ T-cells are the primary targets of HIV , whereaset al. showed a prominent trend towards downregulation of miRNA in HIV+ individuals, with 59 of 62 DE miRNAs downregulated in HIV+ PBMCs [Previous studies have shown unique and variable global modifications in microRNA expressions exhibited by different host cell types and cell lines in response to HIV-1 infection ,15,16,17in vitro-infected PBMCs [in vivo interaction between cell types and other combinatorial interactions, which together play a functional role in determining the dysregulation of host miRNAs during HIV infection. Thus, the analysis of primary CD4+ and CD8+ T-cells we have performed from diverse HIV disease groups carry considerable functional relevance in the context of HIV infection in vivo.A large body of work on miRNAs has been done on cell lines rather than primary patient cells. It is important to iterate that miRNA signatures of PBMCs from HIV+ patient blood are more than 50% discordant with those of ed PBMCs . This imp-value cut-off of <0.05 throughout. The Benjamini–Hochberg algorithm is designed to sort through a list of p-values and determine the likelihood of a false rejection of the null hypothesis  and so “adjust” the p-value to control the likelihood of the false positive. Employing this algorithm provided data of greater integrity, and this is the data we have made use of in interpreting our DE dataset. Due to the stringency of this method of analysis, we failed to detect differential expression of miRNAs in some comparisons, while the DE miRNAs detected for other groups were statistically strong. To clarify any possible occlusion of biologically relevant miRNAs, we also analyzed the dataset without FDR corrective testing. To minimize false positives in the absence of FDR correction, the p-value cut-off was then set at <0.01. This allowed the detection of other DE miRNAs for the groups we failed to detect DE miRNAs using FDR corrective testing. Our hypothesis for using this rationale was that the statistical stringency possibly occludes biologically relevant miRNAs, as several of these miRNAs identified at p < 0.01 are not only of functional relevance, but are also known in the literature.To obtain differentially and statistically significant miRNA candidates, we employed the Benjamini–Hochberg FDR corrective testing with a et al. on PBMCs, where the miRNA signatures of LTNP HIV+ individuals with low viral load and good CD4+ T-cell counts showed greater similarities to uninfected controls than HAART-treated HIV+ patients.Through the use of PCA, we demonstrated that the miRNA profiles of HIV+ CD4+ and CD8+ T-cells were distinct from T-cells of HIV– individuals as all HIV-infected groups  were more closely related as a group and segregated from the uninfected group. PCA also highlighted that the individual samples from the same disease stages cluster together, and the sample classes with the greater similarities were more closely associated. Despite the close relationship of all HIV+ samples, PCA could also distinguish between HIV disease sub-groups, with the LTNP, aviremic, and HIV groups showing a common miRNA signature specific to them. This is plausible, as these three groups have undetectable levels of plasma viremia. Our results can be likened to the results shown by Witwer et al. showed that HIV+ patients could be categorized into disease groups based on DE miRNAs present in the PBMC fraction of their blood. Further, the miRNA signature of CD4+ T-cells from multiply exposed uninfected (MEU) individuals can distinguish them from therapy-naïve infected CD4+ T-cells [et al. also showed that miRNA signatures of LTNPs, MEUs, and therapy-naïve HIV+ patient CD4+ T-cells cluster based on their exposure level and disease state [et al. showed that, through examination of DE miRNAs, LTNPs with low CD4+ T-cell counts and high plasma viremia correlate more closely with HIV+ viremic individuals, and those with high CD4+ T-cell counts correlate with HIV− controls [In support of these findings, several previous papers have suggested that a broad statistical analysis of the miRNAs that are differentially expressed in HIV+ individuals distinguish patients according to their CD4+ T-cell counts, viremia, or HIV exposure level ,9,15. InWhen DE miRNAs were analyzed in the CD8+ T-cells of HIV+ individuals from each of the disease groups , it was shown that some miRNAs were highly specific for these disease groups when compared with negative controls. Namely, miR-1323, -541, and -570 were associated with the CD8+ viremic group, miR-1298 was associated with the CD8+ aviremic group, and the miRNA-572, -126, 151-5p, -486-5p, and -885-3p with the CD8+ LTNP group. This is the first direct evidence of HIV disease-stage-specific miRNAs in CD8+ T-cells, which is consistent with the role of CD8+ T-cells in containing viremia. A clear segregation of DE miRNA in each comparison demonstrates that the CD8+ T-cells are better suited to predicting HIV disease stage and cell-specific miRNAs than their CD4+ T-cell counterparts.Our analysis performed in CD4+ and CD8+ T-cell types suggests that miRNAs-199a-5p, -1280, and -126 were differentially expressed in the CD4+ T-cells of LTNPs when compared to negative controls, and the miRNAs-151-5p, -486-5p, -572, -885-3p, and -126 were differentially expressed in the CD8+ T-cells of LTNPs when compared to negative controls. MiR-126 was differentially expressed in both CD4+ and CD8+ T-cells in the LTNP disease state. Consistent expression trends between microarray and qRT-PCR for miR-126 suggests that this miRNA is significantly downregulated in both the CD4+ and CD8+ T-cells of LTNPs, suggesting that it is a possible biomarker for non-progressive HIV disease. This, to our knowledge, is the first report of a miRNA that correlates with the LTNP disease state and may be involved in the regulation of cellular immunity and possibly the natural control of HIV in LTNPs.vs. negative disease group than in either the viremic or aviremic CD4+ T-cell comparisons against HIV− individuals. Thus, it is likely that miR-199a-5p plays a role in predicting HIV+ status based on CD4+ T-cells. We hypothesize that, although it is expressed and downregulated in the CD4+ T-cells of all HIV+ groups, the degree of downregulation of miR199a-5p in different groups imparts susceptibility or protection, as evident from the CD4+ T-cells from the LTNPs that showed the highest degree of downmodulation of miRNA199-5p, which may play a protective role against the virus. Supporting this conclusion, it has previously been shown that miR-199a  is differentially expressed in the PBMCs of class 1  individuals, which agrees with our work with miRNA 199a showing the strongest downregulation in the LTNPs group. Our analysis using three phenotypically different HIV+ groups clarifies and unambiguously demonstrates a clear association of miR-199a with HIV positivity based uniquely on CD4+ T-cells, a trend which was further confirmed by qRT-PCR analysis, implicating miR-199a to be an HIV-specific miRNA.MiR-199a-5p was differentially expressed in HIV+ CD4+ T-cells belonging to all HIV+ groups analyzed . Stronger downregulation was observed in LTNP et al. and Witwer et al.About other differentially expressed miRNAs, miRNA-155 in HIV+ CD4+ T-cells was recently observed to distinguish LTNPs from therapy-naive HIV+ patients [The 3′ ends of HIV-1 mRNAs (Nef) are targeted by cellular miR-125b , all of which are enriched in resting CD4+ T-cells compared to active CD4+ T-cells . MiR-125et al., and we concur that miR-125 family members are potential candidates associated with latency in CD4+ T-cells [p-values of miRNA candidates with the disease stage added reliability and confidence to these conclusions.Interestingly, our results show that the family of miR-125 was consistently upregulated in the comparison of diseased CD4+ T- and CD8+ T-cells using a non-FDR statistical analysis. This upregulation was markedly higher in the CD4+ T-cells of LTNPs than those of viremic or aviremic individuals, followed by its upregulation in aviremic individuals, suggesting a possible role for this family of miRNAs in aviremia and natural control and possibly viral latency. Our results agree in part with the findings by Huang miR-29 targets the viral Nef 3′UTR and downregulates Nef protein expression and interferes with HIV-1 replication, thereby inhibiting viral production and infectivity in HIV-infected T lymphocytes . Low levAlthough our results did not immediately suggest a role for miR-29 in managing viremia and disease progression, it became clear upon examination that miR-29 was indeed upregulated in aviremic CD4+ T-cells, when compared to both viremic and LTNP CD4+ T-cells. Although it is peculiar that the miR-29s did not show association with the LTNP disease state, this is not unexpected. The biggest difference between HIV+ aviremic and LTNP individuals is the preservation of immune system strength and natural control of HIV in LTNP individuals, where aviremic individuals require HAART to control HIV replication. It is likely that miRNAs may have some role in maintaining below detectable levels of plasma viremia while on HAART therapy. On another note, it is important to iterate that we also found miR-1298 specific to the CD8+ aviremic group (with respect to negative control), showing 2-fold downregulation. This was obtained using the FDR corrective testing, and we believe it is the first miRNA specific to CD8+ T-cells of HIV patients that may be involved in control HIV during HAART therapy and may offer some biological significance. Thus, these two miRNAs may have some functional relevance during HAART therapy, and whether they come into play when therapy is effective in controlling plasma viremia remains unknown. Nonetheless, the two miRNAs in some ways predict the efficacy of HAART and should be explored in the context.Similarly, in aviremic CD8+ T-cells, miR-29b/c were upregulated when compared to negative controls, whereas the miR-29a/b/c were upregulated in aviremic individuals when compared to LTNPs. Taken together, these data suggest that the expression of the cellular miR-29 family is triggered by viral replication and implies a potential role for miR-29 family members in both CD4+ and CD8+ T-cell subsets in HIV infection, although perhaps not in the management of viremia in the LTNP group. Determining whether miR-1298 and the miR-29 family have some functional synergy between them requires future functional validation.miR-17/20 target 3′UTR of PCAF acetyltransferase and are known to indirectly inhibit HIV infection . It has We found that miR-17 and miR-20a were both downregulated in the CD8+ T-cells of LTNPs when compared directly with the CD8+ T-cells from aviremic patients. MiR-17 was downregulated in CD8+ T-cells from the LTNPs when compared against the viremic patients. This upregulation of miRNAs could be related to the inhibition of HIV-1 in a more severe disease state (aviremic on HAART compared to LTNPs), rather than the downregulation of these miRNAs by HIV.p < 0.05) and non-FDR (p < 0.01) statistical analysis, we were able to observe both the strongly DE miRNAs responsible for clear segregation of disease groups using FDR analysis, as well as the gentler trends of miRNA expression between disease classes and cell types in the non-FDR analysis. Our observations regarding the two datasets raise two possibilities: that either most studies in the miRNA field are not as stringently controlled as our primary FDR analysis, or that the stringency we have employed in deriving significantly DE miRNA is too strict. If this is the case, high stringency analysis may occlude biologically significant miRNA candidates, as was apparent when functionally important miRNA deemed important in the literature were confirmed and supported by our non-FDR dataset.As can be noted in several sections of the discussion, the correlation of DE miRNAs observed in our study could only be compared with other studies when non-FDR corrective testing was used. Thus, it is important to reiterate that, since we performed a bimodal analysis of our dataset using FDR  and layered carefully onto 15 mL of Ficoll  in 50-mL tubes, without disrupting the Ficoll border. Separation of the PBMC buffy coat was achieved via centrifugation at 1800 rpm for 20 min at room temperature. The buffy coat containing peripheral blood mononuclear cells (PBMCs) was extracted to another 50-mL tube via transfer pipette. The separated PBMCs were washed twice with DPBS by centrifugation at 1500 rpm for 7 min at 4 °C, then once in 10 mL of cold (4 °C) magnet activated cell sorting method (MACS) buffer , 0.5% FBS  via centrifugation at 1200 rpm for 10 min at 4 °C. The supernatant was aspirated retaining the PBMC pellet.PBMCs from the LTNPs used in this study were previously cryopreserved. These cells frozen in liquid nitrogen were raised to −80 °C for one day before revival. Cells were slowly thawed on ice and were transferred using a transfer pipette into 15-mL tubes containing 5 mL of RPMI 1640 (Lonza BioWhittaker) 10% FBS in 15-mL tubes. The cell suspension was centrifuged for 10 min at 1000 rpm at room temperature to pellet the revived cells. The supernatant was aspirated and decanted retaining the clean cell pellet in preparation for the CD4+ and CD8+ cell separation.g for 10 min at 4 °C. Cells were washed with PBS, and MACS® Column Technology was used for separating PBMCs into different cell subsets. Using MACS® MicroBeads, CD4+ helper T-cells and CD8+ cytotoxic T-cells were extracted according to the manufacturer’s specifications using the positive selection MACS, Miltenyi Biotech . Flow cytometric verification of cell purity of CD4+ and CD8+ T-cells was evaluated using flow cytometry, as previously described by us .The PBMCs were centrifuged at 1000g to remove cell debris. The cell pellet was further washed twice with phosphate buffered saline (PBS) to remove excess antigen. After aspirating all of the PBS, the cell pellet was further washed with 10ml of MACS buffer  + 200 µL of 2 mM EDTA) by centrifuging at 300 Both total RNA and miRNA were extracted from the brain cortex using the miRNeasy Mini Kit  with an integrated step of on-column DNase digestion as per the manufacturer’s protocol for the kit.RNA quality was checked by RNA micro-electrophoresis on a 2100 Bioanalyzer apparatus  and by using the RNA 6000 Nano Labchip Technology Kit. All reagents were equilibrated to room temperature for 30 min before use, and the quantity and quality of RNA were assessed based on the RNA integration numbers (RIN) for each sample. All RNA samples used in q-PCR and miRNA analysis had an RIN of >5, and anything <5 was deemed inappropriate for the miRNA chip, but were considered for the q-PCR.® Biotin HSR RNA Labeling Kit for Affymetrix® GeneChip® miRNA Arrays . miRNA array preparation was conducted according to instructions provided by Genisphere for the Affymetrix GeneChip miRNA array procedure (FlashTag Biotin HSR RNA Labeling Kit for Affymetrix GeneChip miRNA Arrays), including the use of reagents from this kit . Hybridization of prepared samples, incubation, washing, staining, and scanning was completed by the sample submission service at the Ramaciotti Center for Gene Function Analysis, University of New South Wales, Sydney.MicroRNA was prepared for Affymetrix GeneChip arrays using the FlashTagPatients were classed into disease groups based on their HIV plasma VL determined by M3 Prep/Cobas TaqMan HIV-Version 2 kit  to test HIV plasma VL, at the Westmead Hospital ICPMR . Diverse HIV disease groups were analyzed, including HAART-experienced viremic (VL above 40 copies/mL plasma) and aviremic (below detectable levels of plasma viremia) patients. An LTNP group, comprising of HIV+ therapy-naïve individuals who showed high CD4+ and CD8+ T-cell counts and below detectable plasma viremia in the absence of HAART, was also used. All comparisons between disease groups were made against HIV– healthy individuals. Thus, the main criterion for analysis was the stratification of patients into viremic, aviremic, LTNP, and HIV– healthy groups, with the aim of defining disease-stage-specific and cell-specific (CD4+ and CD8+ T-cells) miRNA.2 transformation, and median polish summarization of probesets). The quality of the miRNA dataset and its integrity was assessed using PCA. PCA is a mathematical operation involving applied linear algebra, which transforms datasets into a new coordinate system such that the most variable components of the dataset are presented on axes where each component is independent of the last. This allows a complex dataset to be simplified and visualized by its most important gradients. In turn, the analysis also allows the determination of which factors are the most important in distinguishing one cluster from another. PCA plots were generated, to first test the integrity of infected samples in comparison to uninfected samples, and then PCA was used to inter-compare the disease states for each cell type and the cell types for each disease state  at each point in the list. In this way, it develops a list of corrected or adjusted p-values for the detected differentially expressed miRNAs. A fold change of 2 (up- or downregulation) was also applied to deem any miRNA to be biologically and statistically significant. Although not a part of the result section, data were also processed without FDR corrective testing to see the difference between two datasets. This was to determine whether the statistical manipulation of the data using the FDR method occluded biologically or functionally significant miRNAs. For non-FDR data, a stricter p < 0.01 significance threshold was applied for significant changes, along with the fold change of 2, to ensure biological significance. This method will herein be referred to as the non-FDR method and is used as a comparative set of data for the FDR results.Samples were analyzed using the Partek Genomics Suite software, applying two forms of corrective and statistical testing. Primarily, the analysis of differential expression was completed using Notably, even though non-FDR testing showed a higher number of DE miRNAs than FDR-controlled corrective testing, all microRNAs deemed significant using strict FDR corrective testing were also picked up as highly significant miRNAs in the less stringent, non-FDR method, confirming the reliability of FDR corrective testing.Comparisons of miRNA expression were made in an organized and structured manner. First, differential expression of miRNAs between HIV+ and HIV– individuals was considered. Then, comparisons were made between CD4+ and CD8+ T-cells before examining the differential expression between disease states within CD4+ and CD8+ T-cells. Lastly, results observed in independent CD4+ and CD8+ T-cell analyses were inter-compared. Analyses included clustering, the generation of heat maps, and intergroup comparisons through the generation of Venn diagrams.p-values, fold changes, and differential expression. cDNA was generated from RNA samples using the Qiagen miScript Reverse Transcription Kit . A master mix containing 160 µL of buffer RT and 40 µL of miScript reverse transcriptase mix was produced, and 5 µL was added to each sample of 250-ng RNA. The samples were made with RNase free water (up to 20 µL) and incubated at 37 °C for 60 min, and then 95 °C for 5 min to inactivate the RT enzyme using the Hybaid Thermal Cycler for PCR. Expression levels of two differentially expressed miRNAs (hsa-miR-199a and miR-126) were validated by qRT-PCR using the Qiagen miScript PCR system . Master mixes containing SYBR green PCR master-mix (10 µL/assay), miScript universal primer (2 µL/assay), and RNase-free water (5 µL/assay) was produced for each specific primer. Unique miScript primers specific for the miRNAs of interest were added (2 µL/assay). Data was analyzed using Microsoft Excel. RNU6B control was used to normalize data. All the experiments were performed in duplicate, and relative expression levels of these miRNAs were determined by the ΔΔ−CT method  based on their adjusted MicroRNAs are the master regulators of gene expression; therefore, this analysis of differentially expressed miRNA in CD4+ and CD8+ T-cells at different stages of HIV infection provides highly valuable data on how these two T-cell subsets interact during HIV infection. Our analysis provides data on how miRNAs may guide disease progression: leading to gradual disruption and decline of the immune system in viremic individuals or providing possible resistance against HIV infection, as seen in LTNPs.Determining the role of differentially expressed miRNAs in HIV infection is essential. We believe that an understanding of the dysregulated miRNAs in HIV infection will prove crucial for the development of miRNA-based therapies or the targeting of cellular miRNA as treatment for HIV infection. Before this is possible, a clear understanding of miRNA gene targets is needed. This is a challenge, as one miRNA can target thousands of genes, and HIV infection of cells can induce large changes in gene expression and overall cell state, meaning that the relationships between miRNAs and mRNAs in HIV infection may be very complex. As such, more work is needed to map precisely the mRNA and miRNA interactions that guide HIV pathogenesis. Such analysis will not only allow the development of new strategies for HIV control but may also lead to the identification of miRNA candidates that can be used as a new generation of therapeutic targets to control HIV. For example, a miR-122 agonist, miravirsen, is the first miRNA-targeted drug to be developed for the treatment of viral accumulation in hepatocytes during HCV infection .With an increase of research in the area of miRNA expression profiling and functional analysis, several confounding factors have emerged. Currently, a variety of papers in this area all used different cell types, different definitions of HIV disease states, different methods of analysis and statistical evaluation, and diverse microarray platforms ,15,21,23


Based on our results we propose that amorphous polyP, stabilized onto a collagen matrix, might be a promising component of functionally-active barrier membranes for guided tissue regeneration in medicine and dentistry. We describe a novel regeneratively-active barrier membrane which consists of a durable electrospun poly(ε-caprolactone) (PCL) net covered with a morphogenetically-active biohybrid material composed of collagen and inorganic polyphosphate (polyP). The patch-like fibrous collagen structures are decorated with small amorphous polyP nanoparticles (50 nm) formed by precipitation of this energy-rich and enzyme-degradable  polymer in the presence of calcium ions. The fabricated PCL-polyP/collagen hybrid mats are characterized by advantageous biomechanical properties, such as enhanced flexibility and stretchability with almost unaltered tensile strength of the PCL net. The polyP/collagen material promotes the attachment and increases the viability/metabolic activity of human mesenchymal stem cells compared to cells grown on non-coated mats. The gene expression studies revealed that cells, growing onto polyP/collagen coated mats show a significantly (two-fold) higher upregulation of the steady-state-expression of the  Barrier membranes are required in oral and periodontal surgery to separate rapidly proliferating and regenerating tissue layer(s), like epithelia, from more slowly-growing tissue, e.g., bone . Such anN,N-dimethylformamide or acetone-dichloromethane  (PLA) and integrated Ca-polyP microparticles into the fibers by the mixing of PLA, dissolved in chloroform, and the particles prior to electrospinning. PLA is a slowly-degrading polymer with a half-life in the body of greater than two years [Here we report on a new type of barrier membrane, which consists of a durable organic net covered with a morphogenetically-active hybrid material, consisting of polyphosphate (polyP) and collagen. The durable net was prepared by electrospinning using poly(ε-caprolactone) (PCL), dissolved in organic solvents, like tetrahydrofuran/e e.g., ,8). In a. In aN,Nwo years . It has wo years , which iwo years , but is wo years  with a two years ; the PCLwo years . Recently the biologizing of the inert durable nets was successfully advanced. In this line the mats had been modified along with the sandwich osteotomy procedure by application and inclusion of the recombinant human bone morphogenetic protein-2 (rhBMP-2) into the restoration system . In cont2+. In this hybrid organization polyP retains the characteristic physiological functions  to aiewed in . The woue repair . As a durable scaffold in the mats the polymer PCL was used for electrospinning A. We fabA detailed documentation of the morphology of the PCL mats without, or coated with, polyP/collagen is given in 2 were analyzed by X-ray powder diffraction and found to elicit no distinct sharp peak, indicating that this hybrid material is amorphous (data not shown).The polyP/collagen particles prepared by precipitation with CaClThe biomechanical properties of a non-coated A,B and pStress-strain curves of the dry non-coated PCL-membranes showed a sharp peak, which indicates a fast load increase with very little elongation and, after rupture, also a very fast load reduction A. The ulTo test the stability of the mats in the cell culture assay they were cut into 16 mm discs, inserted into the 15.6 mm large wells and submersed with 2.5 mL medium/serum (a-MEM/FCS). Inspection of the mats at day 5 of incubation revealed that the morphology of the fibers and their arrangement in the mats remained unchanged if compared to day 0 A,B. In cHuman mesenchymal stem cells (hMSCs) were incubated in the 24-well plates onto non-coated and coated PCL mats for five days to determine the properties of the cells to attach to the non-coated or coated PCL mats . The cel450 nm units). During the seven-day incubation the viability increases in assays both with cells growing onto non-coated and polyP/collagen-coated mats. The increase of the viability of cells in assays onto coated mats is significantly higher, compared to those onto non-coated cultures; the absorbance units increase in coated assays to 0.94 ± 0.13 (2.5 doublings). The difference between the two cultures’ conditions is even higher after a 14-day incubation period. The growth rate increase in non-coated cultures is 0.74 ± 0.16 A450 nm units (2.1 doublings) and in coated cultures is 1.34 ± 0.21 A450 nm units (3.6 doublings). hMSCs were seeded onto the mats, either non-coated or polyP/collagen-coated, and incubated for five days in the assay system described under “Material and Methods”. After termination, the viability (metabolic activity) was determined by application of the XTT assay system . At day angiopoietin-2 (ang-2) gene as a marker for differentiation. The data obtained were summarized in GAPDH. The non-coated PCL mats elicit a less stimulatory effect on gene expression. The gene expression status of hMSCs on non-coated and polyP/collagen-coated PCL mats was measured by qRT-PCR using the A bone defect, e.g., after tooth extraction, is preferentially restored by a combination of bone substitute materials which are inserted into the damage and a barrier membrane that is layered on top of it to separate the three-dimensional implant scaffold from the environment, like non-osteogenic soft tissue. These tissue layers are sutured allowing the bone substitution material to be replaced by physiological bone . As outlThe characteristics of the polyP/collagen coating, developed here, is that the hybrid material undergoes degradation within five days of incubation in medium/serum, which allows the dissolution of the nanoparticles from the collagen fibers and themselves to detach from the PCL mats. The rapid dissolution of the polyP/collagen patches is conceivably mediated enzymatically via the alkaline phosphatase, which has been shown to degrade polyP hydrolytically in a progressive manner ,26. Whilangiopoietin-2, a crucial cytokine upregulated during the initial phase of wound healing [To support that the polyP/collagen coated mats elicit morphogenetic activity, which is of advantage for an application as a functionally-active barrier membrane, the steady-state-expression of  healing , was det2+ concentrations, which can be used as grafting material. Tailored as implanted grafts for the filling-in of bone defects , these 1-mm spherical particles were prepared by cryogelation in order to introduce a porous architecture into the hybrid polymer. Now, with the availability of a barrier mat which retains the advantageous properties of polyP/collagen, medical care of the wound, as a whole, can be approached. The clinical application of the developed collagen/polyP-coated electrospun PCL fiber mats are advisable and will be advanced by our group.In conclusion, the data presented here underline the potential successful application of amorphous polyP, stabilized onto a collagen matrix and attached to electrospun PCL fiber mats, as a component in barrier membrane. A more immediate application might be anticipated as a functional barrier membrane for GTR around dental implants . In an aNa-polyphosphate (Na-polyP) with an average chain length of 40 phosphate units was obtained from Chemische Fabrik Budenheim .v/v). The solution obtained was loaded into a plastic syringe with a metallic needle (spinning nozzle). The flow rate was adjusted at 0.1 mL/h by using a syringe pump . The positive output lead of a high voltage supply  was connected with the needle and the negative pole to the collector . The operating voltage was 20 kV over a fixed collection distance of 17 cm. The fibrous mats were collected after 1 h operating/working time. The fibers mats prepared had a thickness of ≈1 mm. They were dried at 40 °C for 24 h to remove the solvent.The PCL mats were fabricated similarly as described before ,34. In b2·2H2O . After one day the mats were removed and dried at room temperature. According to the high porosity and high surface area of the obtained PCL mats, the coating process was done via dipping the mats into a polyP/collagen solution as follows: the collagen suspension (10 mL), containing 0.1 g of collagen  aqueous ethanol solution for 20 min, followed by washing in PBS . Finally the mats were exposed to ultraviolet radiation  for 1 h.Prior to the in vitro experiments the mats were treated with 75%  in a Philips PW 1820 diffractometer  as described previously ,38. A first application of the dental implant and the barrier membrane on a human skull was applied under realistic conditions. The material was provided by the Institute of Functional and Clinical Anatomy, University Medical Center of the Johannes Gutenberg University, Mainz, Germany, according to the ethical guidelines of the University Medical Center Mainz. Digital light microscopy studies were performed with a VHX-600 digital microscope  equipped with a VH-Z25 zoom lens. For the scanning electron microscopic (SEM) visualization a Nova 600 microscope  was used. Reflection electron microscope (REM) was performed in a Philips XL30 microscope  at 15 keV and 21 μA. The mechanical properties of the PCL-polyP/collagen membranes and of the untreated control membranes were determined using a “MultiTest 2.5-xt Force Testing System”, equipped with a 100 N load cell unit . The measurements were performed with standardized stripe specimens (30 × 11 mm) of ~100 μm thickness. Additionally, the final dimensions of the respective samples were determined prior to the measurements by using a sliding caliper. The data were continuously recorded at a frequency of 50 Hz and stored with the “Emperor XT Force software” (Mecmesin Ltd.). For determination of the mechanical properties an aligned tensile force was applied to the samples. The specimens were clamped and pulled apart with a speed of 5 mm/min until rupture. Based on the collected force-displacement-time data the ultimate tensile strength, as well as the maximum elongation, was calculated.2 flasks and cultivated in a-MEM , supplemented with 20% FCS  and 0.5 mg·mL−1 of gentamycin, 100 units mL−1 penicillin, 100 mg·mL−1 of streptomycin, and 1 mM pyruvate . Incubation was performed in an incubator at 37 °C. The assays for cell viability and gene expression studies were started with an inoculum of 3 × 104 cells per well ) in a total volume of 2.5 mL. The cultures were incubated for seven or 14 days, as indicated. The PCL mats, non-coated or polycondensation-coated, were cut into round shapes of 16 mm-large discs and then inserted into the 15.6 mm large wells of the 24 well plates. The discs were fixed to the bottom with a metal ring. Human mesenchymal stem cells (hMSCs) were isolated from bone marrow, obtained from healthy non-diabetic adult volunteers; they were purchased from Lonza Cologne . Incubation was performed as described . The celThe attachment of the cells onto the mats was determined by optical microscopy; the cells were vitally stained with 3 μM Calcein-AM  in PBS . The determination of cell growth/metabolic activity was performed with the colorimetric tetrazolium salt XTT assay , as described . The viaangiopoietin-2 steady-state-expression is considered to be a reliable gene marker for the study of an anabolic effect of a  stimulatory matrix on this process  were selected with Fwd: 5′-GACTTCCAGAGGACGTGGAAAG-3′, and Rev: 5′-CTCATTGCCCAGCCAGTACTC-3′. As a reference, the expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase  Fwd: 5′-ACTTTGTGAAGCTCATTTCCTGGTA-3′ and Rev: 5′-TTGCTGGGGCTGGTGGTCCA-3′ was determined. The qRT-PCRs were performed in an iCycler ; the mean tC values and efficiencies were calculated with the iCycler software (Bio-Rad); the estimated PCR efficiencies range between 93% and 103% [The quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) technique was applied to quantitate the proliferation/differentiation potency of hMSCs cells onto the two different kinds of mats. Wound healing is a complex process; nevertheless, the gene expression profile, especially in hMSCs, with the major emphasis of the iewed in ). In turiewed in . The priand 103% ,44. t-test [After finding that the respective values follow a standard normal Gaussian distribution and that the variances of the respective groups are equal to the standard uncertainty values, expressed as mean ± standard deviation, were estimated. The results were statistically evaluated using the independent two-sample Student’s t-test ,45. The t-test .


However, it was so far not known to what extent bacterial pathogens may contribute to the PET signal. Therefore, we investigated whether clinical isolates of frequently encountered bacterial pathogens take up 18F-FDG in vitro, and whether FDG inhibits bacterial growth as previously shown for 2-deoxy-glucose. 22 isolates of Gram-positive and Gram-negative bacterial pathogens implicated in fever and inflammation were incubated with 18F-FDG and uptake of 18F-FDG was assessed by gamma-counting and µPET imaging. Possible growth inhibition by FDG was assayed with Staphylococcus aureus and the Gram-positive model bacterium Bacillus subtilis. The results show that all tested isolates accumulated 18F-FDG actively. Further, 18F-FDG uptake was hampered in B. subtilis pts mutants impaired in glucose uptake. FDG inhibited growth of S. aureus and B. subtilis only to minor extents, and this effect was abrogated by pts mutations in B. subtilis. These observations imply that bacteria may contribute to the signals observed in FDG-PET infection imaging in vivo. Active bacterial FDG uptake is corroborated by the fact that the B. subtilis phosphotransferase system is needed for 18F-FDG uptake, while pts mutations protect against growth inhibition by FDG.Positron emission tomography (PET) with fluorine-18-fluorodeoxyglucose ( Another major contributor to the problem of bacterial infections is the rapid increase of resistance to multiple antibiotics2. Infections caused by such multidrug-resistant microbes are difficult to treat and responsible for substantial patient morbidity and mortality3.Bacterial infections represent a major problem in hospital and community settings worldwide. In particular, due to increasingly ageing populations, higher rates of organ transplantations and expanding possibilities for chemotherapy, the numbers of immune-compromised patients who are at risk of bacterial infections are steadily increasing. Moreover, this problem is exacerbated by increased use of implanted biomaterials and medical devices, which are particularly susceptible to bacterial contaminations and infection5.The diagnosis of bacterial infections is nowadays mainly based on a combination of several specific and/or non-specific symptoms, systemic inflammation markers and the detection of pathogens by culturing, mass spectrometry, polymerase chain reaction (PCR) or serology. In the ideal situation, the causative agent is identified by culturing, but this takes time and is often challenging since sites of infection may be poorly accessible. Also, there is always a risk of contaminations by other bacteria not related to the actual infection resulting in false-positive culture outcomes. Since these challenges often lead to blind management of bacterial infections, there is an urgent need for diagnostic tools that allow real-time detection of infecting bacteria in the patient, for example by infection imaging99mTechnetium-ciprofloxacin (99mTc-ciprofloxacin Infecton®)6, there are currently no bacteria-specific imaging approaches in clinical use4. A clinically applied alternative is positron emission tomography (PET) with fluorine-18-fluorodeoxyglucose (18F-FDG), which can serve as a diagnostic tool for localizing infections, inflammatory conditions and unknown origins of fever8. In this case, it is generally believed that the infection-specific signal originates from increased glucose uptake by cells involved in the inflammatory process, such as leukocytes, monocytes, lymphocytes, giant cells and macrophages9. Importantly, 18F-FDG-PET is safe, sensitive, easy and rapid to use12, and it is potentially applicable in the monitoring of treatment13. Furthermore, the view that 18F-FDG-PET imaging enables the visualization of bacterial infections and evaluation of antimicrobial therapy has been confirmed in animal experiments and clinical studies18. On the other hand, diagnosing infections with 18F-FDG-PET still remains challenging due to the fact that, for instance, malignancies, sterile inflammation and physiological wound healing, may result in similarly high levels of 18F-FDG uptake as infections17.Although several promising approaches for the direct imaging of infecting bacteria have been proposed and explored, with the exception of 18F-FDG-PET imaging. Specifically, it was not known whether different bacteria can accumulate 18F-FDG, and whether the uptake of FDG is potentially toxic for bacteria as is known to be the case for the structurally related glucose analog 2-deoxy-glucose (2-DG)19. The aims of this study were, therefore, to determine whether clinical isolates of different bacterial pathogens actively take up 18F-FDG in vitro, and whether this may lead to impaired bacterial growth.An intriguing question that has, so far, received little attention is whether infecting bacteria could potentially contribute to the signal derived in 20. 7 bacterial strains from the American Type Culture Collection (ATCC) were also included in the analyses , while all other bacteria were cultured in tryptic soy broth (TSB). All bacteria were grown overnight at 37 °C under constant agitation (250 rpm), with the exception of S. pneumoniae which was grown in standing culture at 5% CO2. The next day, fresh cultures were prepared by dilution of overnight cultures in 9 mL TSB or BHI, and growth was continued up to an optical density (OD) of 1–5 McFarland units. Heat-killed Staphylococcus aureus and Escherichia coli were obtained by incubating the respective cultures for 30 min at 99 °C. To assess 18F-FDG uptake, the bacterial cultures were incubated with 5–10 megabecquerel (MBq) 18F-FDG dissolved in 0.5 mL NaCl (0.9%) at 37 °C for 5 min. Subsequently, 2 mL samples were ‘washed’ twice by centrifugation and resuspension of the bacterial pellets in phosphate buffered saline (PBS). To image 18F-FDG uptake, the bacteria were pelleted by centrifugation, and four Eppendorf tubes containing bacterial pellets were positioned at the center of the ring system of a micro-PET (µPET) Focus 220 scanner  with the bacterial pellets in the field of view. Images were recorded in 30 min and analyzed with the AMIDE software package . To quantify 18F-FDG uptake, the bacterial pellets were placed in a calibrated gamma-counter . The radioactivity in each sample was measured, corrected for background and converted into kilobecquerel (kBq). Each 18F-FDG uptake experiment was performed thrice and all quantifications were executed in quadruple with two different clinical isolates per tested bacterial species. Numbers of colony forming units (CFUs) in different samples were determined in duplicate by resuspension of the bacterial pellets, serial dilution of the resuspended bacteria, and plating on blood agar containing 5% sheep blood. Only in case of Proteus mirabilis, MacConkey agar no.3 plates containing crystal violet were used . CFUs of bacteria not incubated with 18F-FDG were determined as a control to evaluate the effect of 18F-FDG on bacterial survival.For S. aureus and E. coli were grown overnight in TSB as indicated above, and the Bacillus subtilis type strain 168 (Table 18F-FDG (dissolved in 0.5 mL of 0.9% NaCl) was added to the bacterial cultures. Cultures were incubated at 37 °C after the addition of 18F-FDG and culture aliquots of 2 mL were withdrawn at 0, 15, 30, 45 and 60 min. To stop 18F-FDG uptake, each culture aliquot was washed twice by centrifugation and resuspension of the bacterial pellets in PBS. Lastly, the bacteria were pelleted by centrifugation and 18F-FDG uptake in the pellets was measured with a calibrated CompuGamma CS1282 gamma-counter. CFUs were determined at t = 0, t = 30 and t = 60 min by resuspension of the pellets, serial dilution and plating on blood agar as described above. Heat-killed S. aureus and E. coli  served as negative controls. Uptake of 18F-FDG over time was evaluated in duplicate.Clinical isolates of 68 Table  was growS. aureus HG001 and different B. subtilis strains (Table 600) was recorded at 10 min intervals. Each growth experiment was performed at least twice.To assess the toxicity of FDG, the laboratory strain ns Table  were groKruskal Wallis and Mann-Whitney U tests were performed using IBM SPSS Statistics 23. P-values of 0.05 or less were considered significant.18F-FDG, a range of different Gram-positive and Gram-negative bacteria including mostly clinical isolates were incubated with 18F-FDG, and uptake was determined with a calibrated gamma-counter. The 18F-FDG PET signals were quantified and the absorbed activity per CFU was determined as shown in Fig. 18F-FDG uptake. No major differences were observed for most of the investigated Gram-positive and Gram-negative bacteria . Nonetheless, statistically significant differences in 18F-FDG uptake were observed for different bacterial species (p < 0.01) as illustrated in Fig. Streptococcus pyogenes displayed overall the highest uptake activity per 107 CFUs .To evaluate whether bacterial pathogens are capable of active uptake of FUs Fig. . E. coliies Fig. . In cont18F-FDG uptake can also be detected by µPET imaging, the 18F-FDG uptake by several Gram-negative and Gram-positive clinical isolates was qualitatively assessed with a µPET Focus 220 scanner. Indeed, the resulting µPET images clearly show 18F-FDG uptake by the tested bacteria , while E. coli and S. aureus both showed higher signals than the respective heat-killed controls (p < 0.01).To approximate the time needed for uptake of 18F-FDG uptake, we applied the Gram-positive model bacterium B. subtilis for which mutations in the phosphotransferase system (PTS) that facilitates glucose uptake are available22. Indeed, B. subtilis ptsI and ptsG deletion strains displayed a strongly impaired uptake of 18F-FDG compared to the parental wild-type strain B. subtilis 168 , which is in line with the fact that PtsG is the major glucose transporter23, while PtsI is involved in the phosphorylation of internalized glucose making its uptake by PtsG irreversible24. These findings show that, at least in B. subtilis, 18F-FDG is taken up via the PTS system.In order to assess the possible mechanism of S. aureus HG001 and B. subtilis 168, where the growth media were supplemented with increasing concentrations of FDG up to 2 mg/mL. As shown in Fig. S. aureus HG001 reached somewhat lowered OD600 values in the stationary phase when the medium contained 200 µg/mL FDG or more. Similarly, B. subtilis reached a lowered optical density in the stationary phase when cultured in the presence of 2 mg/mL FDG 27. Thus, 2-DG has the ability to de-energize bacteria. This may also be the case for FDG, which would explain the observed growth inhibition at high FDG concentrations. Of note, the growth of B. subtilis ptsG or ptsI deletion mutants was much less affected by FDG than growth of the parental strain, which is consistent with the reduced uptake of 18F-FDG.In line with the finding that 18F-FDG PET is a very useful diagnostic tool for many infectious indications, including fever of unknown origin, endocarditis, spondylodiscitis, and vascular graft infections12. This approach can help in defining the location and extent of infection, thereby providing guidance for biopsy and for therapy follow-up. In general, the detection limit in the imaging of infections by PET will depend on the sensitivity and resolution of the PET camera, on the volume of the bacterial lesion, on the surrounding tissue, and on the target-to-normal tissue (T/N) ratios of radioactivity at sites of infection. Our present study shows that 18F-FDG uptake by infecting bacteria potentially contributes to the overall signal detected by PET imaging of bacterial infections. However, it remains difficult to approximate the contribution of the bacterial 18F-FDG uptake to the total 18F-FDG uptake signal measured during in vivo PET imaging of an infection, because the bacterial contribution to the total signal will vary depending on the bacterial species that causes the infection, on the metabolic state of the infecting bacteria, and on the total number of infecting bacteria. Moreover, the inflammatory status of the infected tissue will impact on the total signal, especially because white blood cell migration may vary depending on the extent of inflammation, and also because inflammation may lead to increased blood flow and vascular permeability. Furthermore, the total signal is likely to depend on the particular body site that is infected. Altogether, this means that, in practice, the relative bacterial contribution to the total 18F-FDG signal will be different for individual patients and for different types of infections. For example, chronic infections can be visualized with 18F-FDG, but the signal contributed by the bacteria themselves may be lower than in acute infections if their metabolic activity is lower, which would result in lower uptake of 18F-FDG. In such cases, an option to improve the bacterial signal could be to start imaging at later time points after tracer injection. Clearly, the conditions in vivo are likely to be different compared to the conditions applied in our in vitro study. It is therefore presently not possible to make reliable calculations of the detection limit for infections in vivo.Currently, there is ample evidence that 18F-FDG PET it is not possible to differentiate between sterile inflammation and infection. Also, 18F-FDG PET will neither tell us whether bacteria are involved in an infection, nor which bacteria are involved. Specific bacteria-targeted PET tracers are therefore desirable. For example, these could include labeled antibiotics like 18F- or 99mTc-ciprofloxacin (Infecton®)31. A very promising alternative for 18F-FDG may be 2-[18 F]-fluorodeoxysorbitol (18F-FDS), since sorbitol is a sugar alcohol that is mainly metabolized by the Gram-negative Enterobacteriaceae25. Another attractive approach appears to be the detection of bacteria with 6-[18F]-fluoromaltose (18F-FM)32, because maltose and maltodextrins are metabolizable by all classes of bacteria, in contrast to mammalian cells. Thus, 18F-FM may allow the distinction between bacterial infection and other causes of inflammation. On this basis it is well conceivable that the combined use of 18F-FDS and 18F-FM will facilitate the detection of a wide spectrum of bacteria causing infections. With the aid of both substrates, it should not only be possible to specifically image infecting bacteria over mammalian cells, but also to discriminate infecting Gram-positive and Gram-negative bacteria. The latter would then guide effective antimicrobial therapy. Yet, despite these evident advantages, no clinical studies on infection imaging with 18F-FDS and 18F-FM have thus far been reported.Lastly, with 18F-FDG is one of the few tracers that may be clinically applied in infection imaging. We now show that this tracer can be taken up by a wide range of commonly encountered bacterial pathogens. We therefore hypothesize that bacterial pathogens may also take up 18F-FDG in vivo, thereby contributing to the FDG-PET signal observed in infection imaging. Future experiments are needed to determine the proportion of 18F-FDG that is taken up by infecting bacteria as compared to the 18F-FDG taken up by inflammatory cells. This will unveil the significance of our present findings in the clinical context.To date,


It is found that the PVDF-HFP hybrid membrane with 2 wt % SiO2 (M3-S2) has better properties and higher filtration rate and separation efficiency for surfactant-stabilized water-in-oil emulsion separation. Moreover, the membrane M3-S2 also exhibits excellent antifouling performance for long-running.Poly(vinylidene fluoride-hexafluoropropylene) (PVDF-HFP) porous membranes are fabricated via thermally induced phase separation (TIPS) with mixed diluent /dioctyl phthalate (DOP)). The effects of mixed diluent are discussed in detail in term of morphology, mean pore size, selective wettability, etc. The results show that the membrane structure changes from spherulitic to bicontinuous with the change of DBP/DOP ratio. It is also found that the degree of crystallization decreases with the decrease of DBP/DOP ratio in mixed diluent. When liquid–liquid (L-L) phase separation precedes solid–liquid (S-L) phase separation, the obtained membranes have outstanding hydrophobicity and lipophilicity, excellent mechanical property. Additionally, the PVDF-HFP hybrid membranes are prepared with silica (SiO Wang et al. [N,N-dimethylformamide (DMF), DMAC and trimethylphosphate (TMP)) on the PVDF-HFP hollow fiber membrane structure and MD performance. Stephan et al. [2 extraction of DBP from PVDF-HFP/DBP former films prepared via TIPS.Recently, an emerging fluoro-copolymer, PVDF-HFP, is attracting more and more attention. As shown in g et al.  preparedg et al.  investign et al. , Hwang en et al.  and Li en et al.  preparedn et al.  fabricatn et al.  preparedn et al.  investigDuring TIPS process, the diluent is a key factor for controlling the membrane structure. The diluent compositions influenced the interaction between the diluent and polymer which have a significant effect on membrane structure . Li et a2 particles and the effects of SiO2 contents on structure and performance are discussed in detail.In this study, PVDF-HFP and mixed diluent (DBP)/(DOP) are used for fabricating porous membranes via TIPS method. The effect of the interaction between mixed diluent and PVDF-HFP on the structure of obtained membranes is investigated and the structure is controlled through changing the composition of mixed diluent. Additionally, PVDF-HFP hybrid membranes are fabricated by blending hydrophobic SiO2 average particle size 40 nm were purchased from Guangzhou GBS High-tech & Industry Co., Ltd.  Graphene (KNG-G5) was purchased from Xiamen Knano Graphene Technology Co., Ltd.  Alcohol, used as an extractant, was provided by AR grade. Diesel was purchased from Tianjin Fengchuan Chemical Reagent Technologies Co. Ltd. .PVDF-HFP , semi-crystalline copolymer, was purchased from Arkema (Singapore). DBP (b.p. 340 °C) and DOP (b.p. 386 °C) were purchased from Tianjin Kermel Chemical Reagent Co. Ltd.  Silica particles . DSC  was used for measuring crystallization temperature (Tc). A small amount of solid mixtures was sealed in an aluminum DSC pan, heated to 180 °C and held for 5 min to erase thermal history and then cooled to room temperature at 10 °C·min−1. During the cooling process, the onset of exothermic peak was identified as Tc.Mixed diluent (DBP and DOP) was mixed with 30 wt % PVDF-HFP at an appropriate temperature (150 °C) in a clean glass vessel and stirred for approximately 3 h to get homogeneous solution. Then, the glass vessel was directly quenched in liquid nitrogen to obtain solid polymer–diluent mixtures. To measure cloud points, a small amount of solid polymer–diluent mixtures was heated to 180 °C on a universal heat-cool stage  using 20 °C·minFirst, 30 wt % PVDF-HFP and 70 wt % mixed diluent were fed into a clean and dried glass vessel and stirred at 150 °C for approximately 4 h to obtain a homogeneous solution. After standing for almost 3 h with atmospheric pressure, the bubble in the solution could be removed. The homogeneous solution was cast onto the pre-clean glass substrate with a thickness of 200 μm at 170 °C and then the glass substrate was directly quenched in the water bath (5 °C). The remaining diluent in films was extracted by absolute ethanol which was then removed by immersing in pure water. Finally, the membranes were dried using a drier . The composition of the obtained PVDF-HFP membranes is exhibited in −1. Five measurements were performed for every sample. The mean pore size of PVDF-HFP porous membranes was investigated using automatic mercury porosimeter  and the porosity of membrane was measured by a gravimetric method and calculated as follows:ε(%) is the porosity of the membrane; 1W and 2W are dry and wet membrane weights, respectively; and Aρ and Bρ are the density of polymer and butanol, respectively.The dried membranes were fractured in liquid nitrogen. The morphologies of membrane surface and cross-section were observed with SEM  after being coated with gold. An atomic force microscope  was employed to analyze the surface states of the prepared membranes. The hydrophobicity and lipophilicity of membranes were measured by contact angle goniometer  at room temperature. To obtain average value, the contact angle was measured five times at different locations. The mechanical strength tests of obtained membranes were performed with an electron instrument . Each membrane was cut into 5 mm (width) × 30 mm (length) specimen and the tensile rate was 20 mm·mincX is the crystallinity of PVDF-HFP membrane, ΔfH is the fusion enthalpy of PVDF-HFP membrane, and Δ−1 [The enthalpy of fusion and degree of crystallization of obtained membranes were measured using DSC  and calculated as follows: .7 J·g−1 .To obtain a homogeneous turbid solution, 1 mL pure water was added dropwise into 100 mL diesel and vigorously stirred for at least 6 h. After standing for 1 day, the surfactant-free water-in-oil emulsion was prepared. For surfactant-stabilized water-in-oil emulsion, 0.2 g span80 with a hydrophile–lipophile balance (HLB) value of 4.3 was added into 100 mL diesel and vigorously stirred for 0.5 h. Then, 1 mL pure water was added dropwise into the mixture and stirred vigorously for 6 h to obtain surfactant-stabilized water-in-oil emulsion. After standing for more than 7 days, no obvious agglomeration and precipitation was observed.J is the filtration rate (L·m−2·h−1), V is the permeate quantity (L), S is the effective area (m2) and t is the testing time (h).The prepared PVDF-HFP membrane was placed on the porous support between feeding bottle and filter tip and sealed carefully by Teflon tape. The separation experiment was carried out at a negative pressure (−0.09 MPa). The filtration rate was measured on the permeated volume of surfactant-free or surfactant-stabilized water-in-oil emulsion through the membrane and determined as follows:fC and pC are water content of feed and permeate water-in-oil emulsion, respectively; and R is the separation efficiency. To get the average value, every membrane was tested at least five times.The separation efficiency of the surfactant-free or surfactant-stabilized water-in-oil emulsion was calculated as follows:The water content in the filtrate was detected by using the Karl Fischer titrator  and then the purity was analyzed to obtain the filter fineness. Photographs of the emulsion and filtrate were taken with a Nikon optical microscope .To evaluate the antifouling performance of the obtained membrane, the filtration rate of the membrane M3-S2 for treatment of emulsions separation in 15 cycles was performed. After each cycle, the membrane was carefully taken out and simply washed with ethanol, and then dried in air.c decreased slowly with the increase of DBP/DOP ratio, while Tcloud decreased rapidly. When the DBP/DOP ratio was larger than 7/3, Tc was higher than Tcloud and the S-L phase separation preceded L-L phase separation. With the further increase of DOP content, the L-L phase separation preceded the S-L phase separation. In addition, the relation between DBP content in mixed diluent was shown in The phase diagram of polymer/diluent system has important data for preparing membranes via TIPS. The phase diagram for this system (30 wt % PVDF-HFP and 70 wt % mixed diluents with varied DBP/DOP ratios) is shown in During the membrane preparation, the kinetic factors and thermodynamic factors are important for membrane structure . The memFrom the surface SEM and AFM images of PVDF-HFP membranes, it can be found clearly that the surface of M0 was composed of spherulites and surface roughness was considerably higher than that of other membranes. As the DBP/DOP ratio decreased (but larger than 7/3), the membrane surface became denser due to the smaller space between spherulites. In addition, the surface became smoother with the decrease of DBP/DOP ratio, which was verified by the AFM images. With further increase of DOP content, the surface became more porous and roughness parameters increased.As discussed above, the ratio of DBP/DOP had an effect on the phase separation process. Therefore, it also influenced the degree of crystallization of PVDF-HFP membrane. The crystallinity was determined by DSC  and the Lipophilic-hydrophobic surface of membrane was a key factor for the water in oil emulsion separation effectively . To inve−2·h−1) than other membranes. The reason is that surfactant-free water-in-oil emulsion passed through the membrane with large pores easily. With the decrease of DBP/DOP ratio, the filtration rate first decreased and then increased. This changing trend has a direct relation with the membrane structure, which first became denser and then looser with decrease of DBP/DOP ratio. Although the filtration rate of membrane M0 was higher than other membranes, the separation efficiency was just 75.51% owing to its larger pores. With the decrease of DBP/DOP ratio, the surface became denser and then more porous, which resulted in the changing trend of filtration rate. From a general view, the highest water rejection in the membrane M3 was acceptable when considering the better hydrophobicity and more applicable pore structure for surfactant-free water-in-oil emulsion. When high separation efficiency is maintained, higher filtration rates mean a membrane is more applicable for surfactant-free water-in-oil emulsion separation. According to the above results, membrane M3 is more applicable for surfactant-free water-in-oil emulsion separation. In addition, some typical properties of the obtained membranes shown in In consideration of lipophilicity and hydrophobicity of membrane, it was promising to separate surfactant-free water-in-oil emulsion. In the surfactant-free water-in-oil emulsion separation experiments, these membranes can repeatedly separate the immiscible surfactant-free water-in-oil emulsion with filtration rate and separation efficiency, as shown in 2 contents and the structure and properties of obtained hybrid membranes were shown in According to −2·h−1. The introduction of SiO2 particles had a significant effect on filtration rate and the filtration rate decreased with the increase of SiO2 contents. The reason is that the introduction of SiO2 particles facilitated the formation of some micro/nano-protrusions on membrane surfaces, and, with the increase of SiO2 contents, more and more microspheres were formed, which blocked the membrane pores. The denser membrane structure suppressed surfactant-stabilized water-in-oil emulsion from passing through the membrane. Compared with the result in In 2 particles, the surface became denser, which resulted in lower filtration rate. However, the separation efficiency was larger than 99% owing to its better hydrophobicity and denser structure. The above results are also shown in Although the filtration of membrane M3-S0 was higher than others, the separation efficiency was just 98.87% owing to its looser surface and relatively poor hydrophobicity. With the addition of SiODuring the long running time, unavoidable membrane fouling occurs, which leads to lower filtration rate and separation efficiency. In this work, a repeating separation experiment is applied to evaluate the antifouling performance of the membrane M3-S2. As shown in cloud of the system increases quickly, while Tc increases slowly with decrease of DBP/DOP ratio. In addition, the main type of phase separation can change from S-L phase separation to L-L phase separation with decrease of DBP/DOP ratio. Therefore, a bicontinuous structure could be obtained by increasing DOP content. In addition, the crystallization of membranes decreases with the decrease of DBP/DOP ratio. The results also show that the membranes prepared with mixed diluent have excellent mechanical properties and hydrophobicity. When the DBP/DOP ratio is 7/3, the obtained membrane reaches optimal properties. In addition, PVDF-HFP hybrid membranes are successfully fabricated with different SiO2 content. The PVDF-HFP hybrid membrane with 2 wt % SiO2 particles shows higher separation efficiency of 99.84% for the surfactant-stabilized water-in-oil emulsion separation. More importantly, the hybrid membrane also exhibits excellent antifouling performance.In this paper, novel PVDF-HFP porous membranes are fabricated via TIPS for oil/water separation. The effects of mixed diluent and additives of the structure and properties are studied. The phase diagram of the ternary system shows that the T